CN111896637A - Detection method of Jinqing intermediate and fingerprint spectrum construction method thereof - Google Patents
Detection method of Jinqing intermediate and fingerprint spectrum construction method thereof Download PDFInfo
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Abstract
The invention discloses a detection method of a cyanine intermediate, which is characterized by comprising the following steps: taking a test sample and a reference substance of the aureonine intermediate for detection, wherein the chromatographic conditions of the detection comprise: octadecylsilane chemically bonded silica is used as a filler; mobile phase: phase B: acetonitrile: methanol (3: 97); phase A: 0.1% H3PO4. The established UPLC content determination and fingerprint analysis method for the Jingqing intermediate of the Gaojinning injection is rapid, convenient and accurate, has good precision, stability and repeatability, can accurately detect the component content of the Jingqing intermediate of the Gaojinning injection, and can objectively evaluate the quality of a sample by the obtained fingerprint.
Description
Technical Field
The invention relates to the technical field of analytical chemistry, in particular to a detection method of a cyanine intermediate and a fingerprint construction method thereof.
Background
For thousands of years, medicines have played irreplaceable roles in guaranteeing and improving the physical health quality of Chinese people. The herbs supplement each other, the herbs pay attention to the quality of the clinical effect, and the qualified herbs are the premise and the basis of the clinical effect. In order to ensure the clinical curative effect of the medicine and the quality of the preparation, the quality of the traditional Chinese medicine or the intermediate thereof is usually controlled by tracing to the source and the intermediate process of the preparation production.
The injection is a pure Chinese medicinal injection prepared from 3 medicinal materials of sweet wormwood, honeysuckle flower and cape jasmine fruit, wherein the sweet wormwood is the monarch drug in the prescription, and has the effects of clearing heat and cooling blood, and dispersing pathogenic wind-heat and pathogenic toxin in the superficial, yin and interior heat. Honeysuckle is a ministerial drug, which excels in clearing heat and removing toxicity, can permeate and disperse exterior evil, and assists the monarch drug sweet wormwood in enhancing the effects of clearing heat and dispersing; gardenia is an adjuvant drug, has the functions of clearing heat and removing toxicity, cooling blood, clearing away heat and relieving restlessness of heart, lung, stomach and triple warmer, and is an assistant drug, namely honeysuckle, for clearing heat and removing toxicity. The combination of the three components can cause the pathogenic wind-heat to be released from the exterior and the pathogenic heat-toxin to be cleared from the interior, thereby achieving the effects of clearing heat, removing toxicity, dispelling wind and relieving exterior syndrome.
The Jinqing intermediate is one of the most important intermediates in the production of the Reduning injection, how to more effectively detect and monitor the quality of the Reduning injection and has direct influence on the quality of a final product.
Disclosure of Invention
In view of the above, the invention aims to provide a UPLC one-test-multiple-evaluation content determination method for multiple components in the aureoblue intermediate, and improve the quality control level of products.
In order to achieve the above object, the present invention provides the following technical solutions:
a detection method of a cyanine intermediate comprises the following steps: taking a test sample and a reference substance of the aureonine intermediate for detection, wherein the chromatographic conditions of the detection comprise:
octadecylsilane chemically bonded silica is used as a filler; mobile phase: phase B: acetonitrile: methanol (3: 97); phase A: 0.1% H3PO4(ii) a The gradient elution was as follows:
and obtaining component information of the aureoblue intermediate or component and content information thereof according to the detection result.
Specifically, the test sample is prepared by precisely weighing 0.06g of the dry extract of the jinqing, placing the dry extract in a 25ml measuring flask, precisely adding 15ml of pure water, carrying out ultrasonic treatment for 60min, cooling, metering to a scale with acetonitrile, shaking up, centrifuging at 12000r/min for 10min, and taking the supernatant as a test sample solution. The preparation method of the Jinqing dry paste optionally comprises the following steps: soaking flos Lonicerae and herba Artemisiae Annuae in 13-18 times of water for 3 hr, heating, decocting, distilling for 2 times, each for 2 hr, and collecting volatile oil. Mixing decoctions, concentrating under reduced pressure to relative density of 1.03-1.08, centrifuging at high speed (20000 rpm) to obtain supernatant, performing fractional ultrafiltration, concentrating ultrafiltrate under reduced pressure to relative density of 1.10-1.12 at 50 deg.C, and vacuum drying to obtain dry extract powder of flos Lonicerae and herba Artemisiae Annuae.
Further, the chromatographic conditions further comprise: detection wavelength: 218-225nm and 320-330 nm; flow rate: 0.28-0.32 ml/min; column temperature: 25-35 ℃.
Further, the chromatographic conditions include: a chromatographic column: agilent Eclipise Plus C18(2.1 mm. times.50 mm, 1.8 μm); mobile phase: phase B: acetonitrile: methanol (3: 97); phase A: 0.1% H3PO4。
Optionally, the column is selected from Waters Xbridge BEH C18(2.1 mm. times.50 mm, 1.7 μm).
Further, the chromatographic conditions further comprise: flow rate: 0.3 ml/min; column temperature: 30 ℃, detection wavelength: 237nm and 324nm (the detection wavelength of iridoid glycoside component is 237nm, and the detection wavelength of organic acid component is 324 nm).
Specifically, the reference substance is selected from one or more of 7-hydroxy swertiamarin, seco-oxyspiroside, neochlorogenic acid, caffeic acid, chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C and total iridoid glycoside.
The invention also provides a fingerprint spectrum construction method of the Jinqing intermediate, which comprises the following steps:
preparation of a test solution: putting the Jinqing dry paste 0.06g into a 25ml measuring flask, precisely adding 15ml of pure water, carrying out ultrasonic treatment for 60min, standing for 1 hour, fixing the volume to the scale with acetonitrile, shaking up, centrifuging at 12000r/min for 10min, and taking the supernatant as a test solution;
performing UPLC detection on the test solution, wherein the UPLC detection chromatographic conditions comprise: octadecylsilane chemically bonded silica is used as a filler; mobile phase: phase B: acetonitrile: methanol (3: 97); phase A: 0.1% H3PO4(ii) a The gradient elution was as follows:
and obtaining the fingerprint of the Jinqing intermediate according to the UPLC detection result.
Further, the chromatographic conditions further comprise: flow rate: 0.28-0.32ml/min, detection wavelength: 218-225nm, column temperature: 25-35 ℃.
Specifically, the method further comprises subjecting a control solution to the UPLC assay; wherein the control is selected from chlorogenic acid. Respectively taking a proper amount of chlorogenic acid reference substances, precisely weighing, placing in a 25ml volumetric flask, dissolving with 25% methanol, and fixing the volume to scale to obtain the reference substance solution.
Further, the chromatographic conditions include: agilent Eclipise Plus C18(2.1 mm. times.50 mm, 1.8 μm); mobile phase: phase B: acetonitrile: methanol (3: 97); phase A: 0.1% H3PO 4; the elution gradient was as follows:
detection wavelength: 220 nm; flow rate: 0.3 ml/min; column temperature: at 30 ℃.
When the method is used for measurement, 1 mu L of each of the reference solution and the test solution can be precisely absorbed and injected into a liquid chromatograph, and the relative retention time and the relative peak area of each chromatographic peak are respectively calculated by taking chlorogenic acid as a reference peak for measurement.
After establishing the map of the Jinqing intermediate according to the method, determining the map of the sample of the Jinqing intermediate to be detected according to the same method, and then comparing the map with the map of the Jinqing intermediate, wherein the similarity is not lower than 0.90.
The invention establishes a UPLC one-test multi-evaluation content determination method for 9 components in the aurantiamarin intermediate, and particularly increases the control standard of 7-hydroxy swertiamarin; meanwhile, a UPLC fingerprint spectrum control method of the intermediate is established, so that the unification with a content measurement and detection method is realized, and the quality control level of the product is effectively improved.
When the method is used for quantitatively detecting the components of the Jingqing intermediate of the Gaojunning injection, the method can comprehensively reflect the chemical components in the Jingqing intermediate of the Gaojunning injection, has the advantages of good separation of various spectral peaks, good peak types and good repeatability, has good linear relation, precision, stability and recovery rate, can accurately detect the component content of the Jingqing intermediate of the Gaojunning injection, and can objectively evaluate the quality of a sample by using an obtained reference fingerprint spectrum.
The established UPLC content determination and fingerprint spectrum analysis method for the Jingqing intermediate of the Gaojunning injection is rapid, convenient and accurate, has good precision, stability and repeatability, can accurately detect the component content of the Jingqing intermediate of the Gaojunning injection, and can objectively evaluate the quality of a sample by the obtained fingerprint spectrum.
Drawings
FIG. 1 is a chromatogram for examining different chromatographic columns in a Jinqing intermediate.
FIG. 2 is a chromatogram for different column temperature investigation of the cyanine intermediate.
FIG. 3 is a chromatogram for different flow rate observations of the aureonine intermediate.
FIG. 4 is a chromatogram obtained by different instrument investigation of the aureonine intermediate.
FIG. 5 is a chromatogram of a cyanine intermediate; wherein, 1, neochlorogenic acid; 2. caffeic acid; 3. chlorogenic acid; 4. cryptochlorogenic acid; 5. 7-hydroxy swertiamarin; 6. cutting semen Strychni Pulveratum glycoside; 7. isochlorogenic acid B; 8. isochlorogenic acid A; 9. isochlorogenic acid C.
FIG. 6 is a comparison fingerprint of a Jinqing intermediate of the Reduning injection; wherein, 1, the chlorogenic acid is new; caffeic acid; s. chlorogenic acid; 3. cryptochlorogenic acid; 4.7-Hydroxyswertiamarin; 5. breaking off the oxidation of the loganin; 6. isochlorogenic acid B; 7. isochlorogenic acid A; 8. isochlorogenic acid C.
Detailed Description
In the invention, the preparation method of the toxic heat removing medicine comprises the following steps:
sweet wormwood 1250g honeysuckle flower 750g cape jasmine 600g
Soaking flos Lonicerae and herba Artemisiae Annuae in 13-18 times of water for 3 hr, heating, decocting, distilling for 2 times, each time for 2 hr, and collecting volatile oil. Mixing decoctions, concentrating under reduced pressure to relative density of 1.03-1.08, centrifuging at high speed (20000 rpm) to obtain supernatant, performing fractional ultrafiltration, concentrating ultrafiltrate under reduced pressure to relative density of 1.10-1.12 at 50 deg.C, and vacuum drying to obtain dry extract powder of flos Lonicerae and herba Artemisiae Annuae. Pulverizing fructus Gardeniae into coarse powder, heating and refluxing with 6 times of 80% ethanol for 1-3 times, each for 1 hr, mixing the medicinal liquids, filtering, recovering ethanol from the filtrate, concentrating to 1:1, adjusting pH to 4.0 with hydrochloric acid, heating at 100 deg.C for 1 hr, refrigerating for 12 hr, filtering, concentrating the filtrate to 50 deg.C relative density of 1.10-1.12, and vacuum drying to obtain fructus Gardeniae dry extract powder. Adding 6g of poloxamer 108 into the volatile oil, grinding, mixing, adding 900ml of water for injection, stirring to clarify, adding the above dried extract powder, stirring to dissolve, adjusting pH to 7.5-8.0 with sodium hydroxide solution, and adding water for injection to 1000 ml. Filtering with G4 sintered funnel, packaging in 10ml ampoule, and sterilizing with 100 deg.C flowing steam. Each 10ml is equivalent to 26 g of crude drug, the injection is instilled into the vein, 10-20ml of the product is injected, and 500ml of 5% grape liquid or 0.9% sodium chloride injection 250-.
In the previous research on the production process of the Gaojunning injection, the control of the extraction process of the Jinqing is mainly characterized in that organic acid components (4 indexes of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid and the like) and iridoid glycoside components represented by seco-oxidized loganin are used as indexes; the quality control of the finished injection of toxic heat removing medicine relates to 11 organic acids and iridoid glycosides, and other trace components such as luteolin and scopoletin. However, no research report about one-test-multiple-evaluation content determination and fingerprint spectrum correlation of the UPLC method for 9 components in the Jinqing dry paste as the intermediate of the Gangning injection is available.
In view of the above, the invention provides a UPLC quantitative detection method for a Qing intermediate component of a Reduning injection and a fingerprint construction method thereof, and a person skilled in the art can appropriately improve process parameters for realization by referring to the contents. It is expressly intended that all such alterations and modifications which are obvious to those skilled in the art are deemed to be incorporated herein. While the method and application of the present invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the method and application described herein may be made and equivalents employed without departing from the spirit and scope of the invention.
Except for special points, the medicines, reagents and instruments used in the technical scheme provided by the invention can be purchased from conventional channels or markets.
Screening of content determination method
1. Correction factor investigation
Taking mixed reference substance solutions with different concentrations, and respectively inspecting the change conditions of the correction factors under different column temperatures, flow rates, chromatographic columns, detection wavelengths, mobile phase compositions and different pH values.
TABLE 1 durability test of correction factor for each compound in the aureonine intermediate
Note: column 1 and column 2 are different batches of AglientZORBAX eclipse C18(2.1 mm. times.50 mm, 1.8 μm) chromatography columns
The preparation method of the mixed reference substance comprises the following steps: respectively weighing appropriate amount of 7-hydroxy swertiamarin, seco-chioperative morroniside, neochlorogenic acid, caffeic acid, chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid A and isochlorogenic acid C as reference substances, precisely weighing, dissolving with 25% methanol, and fixing volume to scale to obtain each single standard solution. Respectively and precisely absorbing appropriate amount of the above single standard solutions, and respectively preparing into mixed standard mother liquor of 7-hydroxy swertiamarin 0.4mg/ml, geniposide 1.189mg/ml, secologanin 0.200mg/ml, neochlorogenic acid 0.246mg/ml, caffeic acid 0.033mg/ml, chlorogenic acid 0.796mg/ml, cryptochlorogenic acid 0.252mg/ml, isochlorogenic acid B0.251 mg/ml, isochlorogenic acid A0.254 mg/ml and isochlorogenic acid C0.232 mg/ml with 25% methanol.
Wherein, besides influencing factors, other chromatographic conditions are as follows: a chromatographic column: agilent Eclipise Plus C18(2.1 mm. times.50 mm, 1.8 μm); mobile phase: phase B: acetonitrile: methanol (3: 97); phase A: 0.1% H3PO4(ii) a Elution gradient is shown in the table below; detection wavelength: 237nm and 324nm (the detection wavelength of iridoid glycoside component is 237nm, and the detection wavelength of organic acid component is 324 nm); flow rate: 0.3 ml/min; column temperature: at 30 ℃. Gradient elution, conditions were as follows:
the results show that the correction factors have no obvious change under the conditions of different column temperatures, flow rates, chromatographic columns, detection wavelengths, mobile phase compositions and different pH values.
2. Durability examination of chromatographic conditions
(1) Chromatographic column investigation
The separation of 9 fractions from the azure intermediate was compared between 2 different types of columns (column 1: active ZORBAX Eclipse Plus C18(2.1 mm. times.50 mm, 1.8 μm) and column 2: Waters Xbridge BEH C18(2.1 mm. times.50 mm, 1.7 μm)).
TABLE 2 chromatographic column investigation of 9 different components in the Jinqing intermediate
As a result: referring to the chromatographic conditions in the correction factor inspection, the test solution is examined, and fig. 1 and table 2 show that 9 chromatographic peaks in the test solution can be well separated.
(2) Investigation of different elution conditions
The first condition is as follows: a chromatographic column: agilent Eclipse Plus C18(2.1 mm. times.50 mm, 1.8 μm); mobile phase: phase B: methanol; phase A: 0.1% H3PO4(ii) a Elution gradient is shown in the table below; detection wavelength: 237nm and 324 nm; flow rate: 0.25 ml/min; column temperature: 30 ℃;
as a result: some components are not separated well, and further optimization of conditions is required.
And a second condition: a chromatographic column: waters HSS T3C18(3.0 mm. times.100 mm, 1.7 μm); mobile phase: phase B: acetonitrile; phase A: 0.1% H3PO4(ii) a Elution gradient is shown in the table below; detection wavelength: 237nm and 324 nm; flow rate: 0.3 ml/min; column temperature: 30 ℃;
as a result: some components are not separated well, and further optimization of conditions is required.
And (3) carrying out a third condition: a chromatographic column: waters HSS T3C18(3.0 mm. times.100 mm, 1.7 μm); mobile phase: phase B: methanol: acetonitrile (1: 1); phase A: 0.1% H3PO4(ii) a Elution gradient is shown in the table below; detection wavelength: 237nm and 324 nm; flow rate: 0.4 ml/min; column temperature: 30 ℃;
as a result: some components are not separated well, and further optimization of conditions is required.
And a fourth condition: a chromatographic column: agilent Eclipse Plus C18(2.1 mm. times.50 mm, 1.8 μm); mobile phase: phase B: methanol: acetonitrile (3: 97); phase A: 0.1% H3PO4(ii) a Elution gradient is shown in the table below; detection wavelength: 237nm and 324 nm; flow rate: 0.3 ml/min; column temperature: 30 ℃; the results are shown in FIG. 3.
As a result: some components are not separated well, and further optimization of conditions is required.
And a fifth condition: a chromatographic column: agilent Eclipise Plus C18(2.1 mm. times.50 mm, 1.8 μm); mobile phase: phase B: acetonitrile: methanol (3: 97); phase A: 0.1% H3PO4(ii) a The elution gradient is shown in the table below; detection wavelength: 237nm and 324nm (the detection wavelength of iridoid glycoside component is 237nm, and the detection wavelength of organic acid component is 324 nm); flow rate: 0.3 ml/min; column temperature: at 30 ℃.
As a result: as a result, the separation degree of 9 main components in the Jinqing dry paste is better under the condition, and the detection requirement of a sample is met, so that the Jinqing dry paste is used as a final detection condition.
(3) Investigation of different column temperatures
The separation of 9 components in the aureocyanine intermediate was compared at different column temperatures (25 ℃, 30 ℃, 35 ℃) with reference to the chromatographic conditions in "correction factor review".
TABLE 3 investigation of different column temperatures for 9 components in the Jinqing intermediate
Fig. 2 and table 3 show that too high or too low column temperature affects the separation degree of 9 components in the interior of the.
(4) Investigation of different flow rates
Referring to the chromatographic conditions in the "correction factor examination", the separation of 9 components in the injection of Reduning was compared at different flow rates (0.28ml/min, 0.30ml/min, 0.32 ml/min).
TABLE 4 investigation of different flow rates of 9 components in the aureonine intermediate
FIG. 3 and Table 4 show that 9 components in the injection of Reduning were detected well at flow rates of 0.28ml/min, 0.30ml/min, and 0.32 ml/min.
(5) Investigation of different instruments
Apparatus 1: agent 1290Infinity II (binary Pump, DAD Detector)
An instrument 2: agent 1260Infinity II (binary Pump, DAD Detector)
The separation of 9 components from the intermediate of aureocyanine under different instrument conditions was compared with reference to the chromatographic conditions in "correction factor review".
TABLE 5 Instrument investigation of 9 different Components in the Jinqing intermediate
Fig. 4 and table 5 show that both apparatus 1 and apparatus 2 are suitable for the separation of 9 components of the auraptene intermediate.
Quantitative detection method for components of Jinqing intermediate of Reduning injection and methodology investigation thereof
Instruments and reagents:
the instrument comprises the following steps: agilent 1290 high performance liquid chromatograph (Agilent technologies, ltd.); ME204E electronic analytical balance (mettler-toledo shanghai ltd); an XS205 electronic analytical balance (Mettler-Torledo Shanghai Co., Ltd.); KH-300DB ultrasonic cleaner (Kunshan Selengensis ultrasonic Instrument Co., Ltd.).
Reagent: methanol (Nanjing chemical reagents, Inc., lot number 180522285K); acetonitrile (Tedia, batch No. 18035148); methanol (Tedia, batch No. 17095086); phosphoric acid (Nanjing chemical reagents, Inc., batch No.: 170825965E).
The medicinal materials are as follows: the Jinqing intermediate (Jiangsu Kangyuan pharmaceutical Co., Ltd., batch No. 160322-; chlorogenic acid (China institute for testing and testing food and drug, batch No. 110753-201716, purity 99.3%) Thermodulin injection (Jiangsu Kangyuan pharmaceutical industry Co., Ltd., batch No. 160706-160715); geniposide (China institute for testing and testing food and drug; batch No. 110749) -201617, purity: 97.6%); secologanin (Dode limited company, lot No. 17033103, purity: 98.5%); neochlorogenic acid (Shanghai-sourced leaf Biotechnology Co., Ltd., batch No.: P13O8F45676, purity: 99.6%); caffeic acid (China institute for testing and testing food and drug; batch No. 110885) and 201703, purity: 99.7%); chlorogenic acid (China institute for testing food and drug, batch No. 110753-one 201716, purity: 99.3%); cryptochlorogenic acid (Shanghai leaf Biotech Co., Ltd., batch No: Y12O8H45678, purity: 98.5%); isochlorogenic acid A (China institute for testing and testing food and drug, batch No. 111782-201706, purity: 97.3%); isochlorogenic acid B (Shanghai Yuan leaf Biotech Co., Ltd., batch No. P20J9F53191, purity: 97.3%); isochlorogenic acid C (China institute for testing and testing food and drug, lot number: 111894-201001, purity: 89.6%); 7-Hydroxyswertiamarin (Shanghai Shidande Standard technical services Co., Ltd., lot number: 5685, purity: 96.78%).
1. UPLC quantitative detection method for components of Jinqing intermediate of Reduning injection
(1) Preparation of a test solution: precisely weighing 0.06g of the azurin intermediate, placing the azurin intermediate in a 25ml measuring flask, precisely adding 15ml of pure water, carrying out ultrasonic treatment for 60min, cooling, fixing the volume to a scale with acetonitrile, shaking up, centrifuging at 12000r/min for 10min, and taking supernatant as a test solution.
(2) Preparation of control solutions: preparing mixed standard mother liquor: respectively weighing appropriate amount of geniposide, secologanin, neochlorogenic acid, caffeic acid, chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, and isochlorogenic acid C as reference substances, precisely weighing, dissolving with 25% methanol, and fixing volume to scale to obtain each single standard solution. Respectively and precisely absorbing appropriate amount of the above single standard solutions, and respectively preparing into mixed standard mother liquor of gardenoside 1.189mg/ml, 7-hydroxy swertiamarin 0.4mg/ml, secologenin 0.200mg/ml, neochlorogenic acid 0.246mg/ml, caffeic acid 0.033mg/ml, chlorogenic acid 0.796mg/ml, cryptochlorogenic acid 0.252mg/ml, isochlorogenic acid B0.251 mg/ml, isochlorogenic acid A0.254 mg/ml and isochlorogenic acid C0.232 mg/ml with 25% methanol.
Preparation of standard working solution 1: taking a proper amount of the reference mother liquor to prepare a mixed reference solution, and putting 0.5ml, 1ml, 2ml and 3ml of the mixed reference mother liquor into a 5ml volumetric flask for setting scales respectively to obtain a series of reference solutions. See table 6.
Preparation of a standard working solution 2: accurately weighing 8.68mg of 7-hydroxy swertiamarin reference substance, placing in a 20ml measuring flask, dissolving with anhydrous methanol, and fixing volume to scale to obtain single-label mother liquor with concentration of 0.420 mg/ml. Respectively taking 1ml, 2ml, 4ml and 6ml of single-standard mother liquor, placing the single-standard mother liquor into a 10ml volumetric flask, and fixing the volume and placing scales to obtain the series of standard working solutions. See table 6.
TABLE 69 concentration information (mg/ml) of the Compounds
(3) Chromatographic conditions are as follows: a chromatographic column: agilent Eclipise Plus C18(2.1 mm. times.50 mm, 1.8 μm); mobile phase: phase B: acetonitrile: methanol (3: 97); phase A: 0.1% H3PO 4; elution gradient is shown in the table below; detection wavelength: 237nm and 324nm (the detection wavelength of iridoid glycoside component is 237nm, and the detection wavelength of organic acid component is 324 nm); flow rate: 0.3 ml/min; column temperature: at 30 ℃. Gradient elution, conditions were as follows:
(4) and (3) determination of a correction factor: the 5 mixed reference solutions with different concentrations in Table 1 were sampled and analyzed, wherein the iridoid glycoside component can be jasminoidin as internal reference, the organic acid component can be chlorogenic acid as internal reference, and the correction factors were calculated respectively, and the results are shown in Table 7.
TABLE 7 correction factor information of each compound in the golden blue dry paste
(5) The determination method comprises the following steps: the control solution and the sample solution were precisely aspirated at 1. mu.L each, and the resulting solution was injected into a liquid chromatograph, and measured using a calibration factor, as shown in FIG. 5.
2. Investigation of linear relationships
Precisely sucking 1 μ l of each of 2 series of reference solutions, injecting into liquid chromatograph, and measuring. And recording peak areas of the chromatographic peaks, drawing a standard curve by taking the injection concentration (mg/ml) as an abscissa (x) and the peak areas as an ordinate (y), and calculating a regression equation, wherein the result is shown in a table 8.
Table 8 linear regression equation for 9 compounds in the aureonine intermediate
3. Precision test
A control solution (BQ4) with the same concentration was sampled continuously for 6 injections, and the peak area was measured to calculate the RSD value of each peak area of the chromatogram, as shown in Table 9. The result shows that the method has good precision.
TABLE 9 precision of 9 compounds in the aureonine intermediate
4. Stability test
A reference solution (BQ3) and a test solution (batch number: 180541) are respectively taken as one portion, injection is carried out for 0, 2, 4, 8, 12 and 24 hours, and the RSD value of the peak area of the chromatographic peak of each component is calculated, which is shown in Table 10. The results show that the method has good stability.
TABLE 10 stability of 9 Compounds in the aureonine intermediate
5. Repeatability test
The same sample batch (batch No. 180541) was used, and 6 parts of the sample solution were prepared in parallel according to the preparation of the sample solution, and the measurement was carried out under the above chromatographic conditions, as shown in Table 11. The results show that the method has good repeatability.
TABLE 11 repeatability (mg/g) of 9 compounds in the aureonine intermediate
6. Sample application recovery test
TABLE 12 sample recovery of 9 compounds from the aureonine intermediate
7. Determination of sample content
TABLE 1310 content of ingredients (mg/g) in batch Jinqing intermediate
Establishment of fingerprint detection method for Jinqing intermediate of Reduning injection
1. Fingerprint spectrum detection method for Qing intermediate of Reduning injection
(1) Preparation of a test solution: precisely weighing 0.06g of the Jinqing dry paste, placing the Jinqing dry paste in a 25ml measuring flask, precisely adding 15ml of pure water, carrying out ultrasonic treatment for 60min, placing for 1 h, fixing the volume to a scale with acetonitrile, shaking up, centrifuging at 12000r/min for 10min, and taking supernatant as a test solution.
(2) Preparation of control solutions: respectively taking a proper amount of chlorogenic acid reference substances, precisely weighing, placing in a 25ml volumetric flask, dissolving with 25% methanol, and fixing the volume to scale.
(3) Chromatographic conditions are as follows: a chromatographic column: agilent Eclipise Plus C18(2.1 mm. times.50 mm, 1.8 μm); mobile phase: phase B: acetonitrile: methanol (3: 97); phase A: 0.1% H3PO 4; elution gradient is shown in the table below; detection wavelength: 220 nm; flow rate: 0.3 ml/min; column temperature: at 30 ℃.
(4) The determination method comprises the following steps: precisely sucking 1 μ L of each of the reference solution and the sample solution, injecting into a liquid chromatograph, and calculating relative retention time and relative peak area of each chromatographic peak with chlorogenic acid as reference peak.
2. Precision test
The sample solution was prepared by the method described in example 1, and 6 needles were continuously injected, and the relative retention time and the relative peak area of each chromatographic peak and the corresponding RSD value were calculated using chlorogenic acid as a reference peak, respectively, and the results are shown in tables 14 and 15. The relative retention time and the relative peak area RSD of each chromatographic peak are both less than 5.0 percent, which indicates that the method has good precision.
TABLE 14 fingerprint Peak relative Retention time precision review
TABLE 15 fingerprint Peak relative Peak area precision
3. Stability test
The test solutions were prepared as described in example 1, and the relative retention times and relative peak areas of the respective chromatographic peaks and the corresponding RSD values were calculated using chlorogenic acid as a reference peak, by feeding samples at 0, 2, 4, 8, 12, and 24 hours, respectively, and the results are shown in tables 16 and 17. The relative retention time of each chromatographic peak and the RSD of the relative peak area are less than 5.0 percent, which indicates that the stability of the Jinqing dry paste is good within 24 hours.
TABLE 16 fingerprint Peak relative Retention time stability Studies
TABLE 17 fingerprint Peak relative Peak area precision investigation
4. Repeatability test
The test solutions were prepared in parallel with 6 portions according to the method described in example 1, and the relative retention times and the relative peak areas of the respective spectral peaks and the corresponding RSD values were calculated using chlorogenic acid as a reference peak, and the results are shown in tables 18 and 19. The relative retention time and the relative peak area RSD of each chromatographic peak are both less than 5.0 percent, which shows that the method has good repeatability.
TABLE 18 fingerprint Peak relative Retention time repeatability test
TABLE 19 repeatability test of relative peak areas of fingerprint peaks
5. Sample assay
10 batches of the Jinqing dry paste are analyzed according to the established method, and the fingerprint spectrum is collected, and the result is shown in tables 20 and 21. The 10 batches of sample chromatograms obtained were introduced into "traditional Chinese medicine chromatogram fingerprint similarity evaluation software 2004A" developed by the ministry of the national pharmacopoeia committee for data processing analysis, a control fingerprint (see fig. 6) was generated, and the similarity was evaluated.
TABLE 2010 batch of dry Jinqing extract relative retention time of fingerprint peaks
TABLE 2110 relative peak area of dry extract fingerprint
The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. The detection method of the aureonine intermediate is characterized by comprising the following steps: taking a test sample and a reference substance of the aureonine intermediate for detection, wherein the chromatographic conditions of the detection comprise:
octadecylsilane chemically bonded silica is used as a filler; mobile phase: phase B: acetonitrile: methanol (3: 97); phase A: 0.1% H3PO4(ii) a The gradient elution was as follows:
and obtaining component information of the aureoblue intermediate or component and content information thereof according to the detection result.
2. The detection method according to claim 1, wherein the sample is prepared by weighing the dried Jinqing paste, placing the weighed Jinqing paste into a measuring flask, adding pure water, carrying out ultrasonic treatment, fixing the volume to a scale with acetonitrile, shaking up, centrifuging at 12000r/min for 10min, and taking the supernatant as a sample solution.
3. The detection method according to claim 1, wherein the chromatographic conditions further comprise: detection wavelength: 218-225nm and 320-330 nm; flow rate: 0.28-0.32 ml/min; column temperature: 25-35 ℃.
4. The detection method according to claim 1, wherein the chromatographic conditions comprise: a chromatographic column: agilent eclipise Plus C18(2.1 mm. times.50 mm, 1.8 μm); mobile phase: phase B: acetonitrile: methanol (3: 97); phase A: 0.1% H3PO4。
5. The detection method according to claim 4, wherein the chromatographic conditions further comprise: flow rate: 0.3 ml/min; column temperature: 30 ℃, detection wavelength: 237nm and 324 nm.
6. The assay of claim 1, wherein the control is selected from one or more of 7-hydroxyswertiamarin, secologanin, neochlorogenic acid, caffeic acid, chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, and total iridoid glycosides.
7. A fingerprint construction method of a Jinqing intermediate is characterized by comprising the following steps:
preparation of a test solution: placing the dried Jinqing paste into a measuring flask, precisely adding pure water, carrying out ultrasonic treatment for 60min, standing for 1 h, fixing the volume to a scale with acetonitrile, shaking up, centrifuging at 12000r/min for 10min, and taking supernatant as a test solution;
detecting the test solution, wherein the detection chromatographic conditions comprise: octadecylsilane chemically bonded silica is used as a filler; mobile phase: phase B: acetonitrile: methanol (3: 97); phase A: 0.1% H3PO4(ii) a The gradient elution was as follows:
and obtaining the fingerprint of the Jinqing intermediate according to the detection result.
8. The method of construction according to claim 7, wherein the chromatographic conditions further comprise: flow rate: 0.28-0.32ml/min, detection wavelength: 218-225nm, column temperature: 25-35 ℃.
9. The method of constructing according to claim 7, further comprising subjecting a control solution to the detecting; wherein the control is selected from chlorogenic acid.
10. The method of construction according to claim 7, wherein the chromatographic conditions comprise: agilent eclipise Plus C18(2.1 mm. times.50 mm, 1.8 μm); mobile phase: phase B: acetonitrile: methanol (3: 97); phase A: 0.1% H3PO4(ii) a The elution gradient was as follows:
detection wavelength: 220 nm; flow rate: 0.3 ml/min; column temperature: at 30 ℃.
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